Heliotropium ramosissimum metabolic profiling, in silico and in vitro evaluation with potent selective cytotoxicity against colorectal carcinoma

Heliotropium is a genus of the Boraginaceae family. Its members are used in many traditional and folklore medicines to treat several ailments. Despite this widespread usage, only a few evidence-based scientific studies investigated and identified its phytoconstituents. Herein, we documented the chemical profile of the Heliotropium ramosissimum methanolic extract using gas chromatography-mass spectrometry (GC–MS) and liquid chromatography-tandem mass spectrometry (LC–ESI–MS/MS) and assessed its antioxidant and cytotoxic effects. The methanolic extract exhibited high phenolic content (179.74 ± 0.58 µg/mL) and high flavonoid content (53.18 ± 0.60 µg/mL). The GC–MS analysis of the lipoidal matter allowed us to identify 41 compounds with high percentages of 1,2-benzenedicarboxylic acid, bis(2-methoxyethyl) ester (23.91%), and 6,10,14-trimethylpentadecan-2-one (18.74%). Thirty-two phytomolecules were tentatively identified from the methanolic extract of H. ramosissimum using LC–MS/MS. These compounds belonged to several phytochemical classes such as phenolic acids, alkaloids, coumarins, and flavonoids. Furthermore, we assessed the antioxidant activity of the methanolic extract by DPPH assay and oxygen radical absorbance capacity assay, which yielded IC50 values of 414.30 µg/mL and 170.03 ± 44.40 µM TE/equivalent, respectively. We also assessed the cytotoxicity of the methanolic extract on seven different cell lines; Colo-205, A-375, HeLa, HepG-2, H-460, and OEC showed that it selectively killed cancer cells with particularly potent cytotoxicity against Colo-205 without affecting normal cells. Further studies revealed that the extract induced apoptosis and/or necrosis on Colo-205 cell line at an IC50 of 18.60 µg/mL. Finally, we conducted molecular docking on the LC–ESI–MS/MS-identified compounds against colon cancer antigen 10 to find potentially cytotoxic compounds. Binding score energy analysis showed that isochlorogenic acid and orientin had the highest affinity for the colon cancer antigen 10 protein, with binding scores of (− 13.2001) and (− 13.5655) kcal/mol, respectively. These findings suggest that Heliotropium ramosissimum contains potent therapeutic candidates for colorectal cancer treatment.

(1 kg) were defatted with n-hexane (3 × 1.5 L) to prepare the lipoidal matter and then extracted with methanol (3 × 1.5 L) to prepare the total methanolic extract used in this study 15 . We performed the extractions in each solvent until exhaustion. After completing the process, we removed each solvent under reduced pressure using a rotary evaporator (Acculab, USA) at 50 °C. The n-hexane yielded 5 g of residue, and the total methanolic extracts weighed 10 g. We stored the extracts in a vacuum desiccator until further use 16,17 . Phytochemical studies. Phytochemical screening. We identified the presence of phytochemical classes in freshly prepared crude extracts of the flowering aerial parts of H. ramosissimum using standard colorimetric procedures 18,19 .
Estimation of the total phenolic and flavonoid contents. The total phenolic content was calculated as gallic acid equivalents (GAE) per g of the sample using the Folin-Ciocalteu reagent and a calibration curve prepared with gallic acid 20 . Moreover, the total flavonoid content was determined as rutin equivalents (RE) per g of the sample using aluminum chloride (AlCl 3 ) colorimetric assay 20 . Metabolomic analysis. GC-MS analysis of the lipoidal matter. The chemical composition of the lipoidal matter of the aerial parts of H. ramosissimum was determined using a Trace GC-TSQ mass spectrometer (Thermo Fisher Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (Thermo Fisher Scientific, Austin, TX, USA) (30 m × 0.25 mm × 0.25 µm film thickness). The initial column oven temperature was 50 °C. It was then increased to 250 °C at 5 °C/min, held for 2 min, increased to the final temperature of 300 °C at 30 °C/ min, and held for 2 min. The injector and MS transfer line were kept at 270 °C and 260 °C, respectively. The carrier gas (helium) had a constant flow rate of 1 mL/min. The solvent delay was 4 min, and diluted 1 µL samples were injected automatically using Autosampler AS1300 coupled with GC in split mode. We collected electron ionization mass spectra at an ionization voltage of 70 eV over the m/z range 50-650 in full scan mode. We set the www.nature.com/scientificreports/ ion source temperature to 200 °C. We identified the components by comparing their mass spectra with those of the WILEY 09 and NIST 14 mass spectral libraries 16 .

LC-ESI-MS/ MS profiling.
We performed the LC-ESI-MS/MS analysis of the methanolic extract on an ExionLC AC system coupled with a SCIEX Triple Quad 5500 + MS/MS system equipped with an electrospray ionization (ESI) system. The samples were eluted on an Ascentis C18 Column (4.6 × 150 mm, 3 µm). Mobile phases consisted of eluent A (0.1% formic acid) and eluent B (acetonitrile, LC grade). The mobile phase gradient was programmed as follows: 10% B at 0-1 min, 10%-90% B at 1-21 min, 90% B at 21-25 min, and 10% at 25.01-28 min. The flow rate was 0.5 mL/min, and the injection volume was 10 µL. MS/MS analysis used positive and negative ionization modes with a scan (EMS-IDA-EPI) from 100 to 1000 Da for MS1 with the following parameters: curtain gas, 25 psi; Ion Spray voltage, 5500 and − 4500 v for positive and negative modes, respectively; source temperature, 500 °C; ion source gas 1 & 2, 45 psi and from 50 to 800 Da for MS2; declustering potential, 80; collision energy, 35 and − 35 for positive and negative modes, respectively; and collision energy spread, 20 21 . We identified the compounds using MS-DIAL software version 4.70.
Free radical scavenging activity assessment by DPPH assay. We prepared 1000 and 100 μg/mL solutions from the methanolic extract to identify a range within which the inhibitory concentration 50 (IC 50 ) lay. We serially diluted the solutions exceeding 50% five times. We prepared a 100 μM Trolox stock solution in methanol. From this stock solution, we prepared 50, 40, 30, 20, 15, 10, and 5 μM solutions. We performed the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical assay as described by Boly et al., 2016 17,20 . Briefly, we added 100 μL of freshly prepared DPPH reagent (0.1% in methanol) to 100 μL of the sample in a 96-well plate (n = 6) that we incubated at room temperature for 30 min in the dark. Next, we measured the reduction in DPPH color intensity at 540 nm using the microplate reader FluoStar Omega. We presented the data as mean ± standard deviation according to the following equation: Assessment of the oxygen radical absorbance capacity (ORAC assay).  www.nature.com/scientificreports/ Cytotoxicity assay. We assessed cell viability through a sulforhodamine B (SRB) assay. We added 100 μL of cell suspension (5 × 10 3 cells) to 96-well plates and incubated them in a complete medium for 24 h. We then treated the cells with 100 μL of medium containing samples at different concentrations (10 and 100 µg/mL). After 72 h of exposure, we fixed the cells by replacing the medium with 150 μL of 10% trichloroacetic acid and incubated them at 4 °C for 1 h. Next, we removed the trichloroacetic acid solution and washed the cells five times with distilled water. We then added 70 μL of SRB solution (0.4% w/v) and incubated the mixture in a dark place at room temperature for 10 min. We washed the plates three times with 1% acetic acid and allowed them to air-dry overnight. Then, we added 150 μL of TRIS (10 mM) to dissolve the protein-bound SRB stain and measured the absorbance at 540 nm using a BMG LABTECH-FLUOstar Omega microplate reader (Ortenberg, Germany) 23 .
Annexin-based apoptosis assay. Colo-205 cells were treated with either doxorubicin (10 µM) as a positive control or the total methanolic extract of H. ramosissimum for 48 h. Next, we collected the cells (10 5 cells) by trypsinization and washed them twice with ice-cold phosphate-buffered saline (PBS, pH 7.4). We then incubated the cells in the dark with 0.5 mL of Annexin V-FITC/propidium iodide (PI) solution for 30 min at room temperature according to the manufacturer's protocol (Annexin V-PI staining apoptosis detection kit from Abcam Inc., Cambridge Science Park, Cambridge, UK). After staining, we injected the cells into an ACEA Novocyte flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA) and detected the FITC and PI fluorescent signals using FL1 and FL2 signal detectors, respectively (λ ex/em = 488/530 nm for FITC and λ ex/em = 535/617 nm for PI).
For each sample, we acquired 12,000 events and quantified the FITC-and PI-positive cells by quadrant analysis using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA) 24 .
Cell cycle distribution analysis. After treating the cells (10 5 cells) with the total methanolic extract of H. ramosissimum for 48 h or paclitaxel (1 µM) for 24 h, as a positive control, we collected them by trypsinization and washed them twice with ice-cold PBS (pH 7.4). We then resuspended the cells in 2 mL of 60% ice-cold ethanol and incubated them at 4 °C for 1 h for fixation. Next, we washed the fixed cells twice with PBS (pH 7.4) and resuspended them in 1 mL of PBS containing 50 µg/mL RNAase A and 10 µg/mL PI. After 20 min of incubation in the dark at 37 °C, we analyzed the cells' DNA contents by flow cytometry using an FL2 (λ ex/em 535/617 nm) signal detector (ACEA Novocyte flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA). For each sample, 12,000 events were acquired. We determined cell cycle distribution using ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA) 25 .

Statistical analysis.
We carried out triplicate experiments and analyzed data using Microsoft Excel. We determined the IC 50 using GraphPad Prism 5 by converting the concentrations to their logarithmic value and using the "non-linear inhibitor regression equation (log (inhibitor) vs. normalized response-variable slope equation)" function 26 .

Molecular docking.
We performed docking studies using Molecular Operating Environment (MOE, 2014.0901) 27 . We retrieved the three-dimensional (3D) structures of serologically defined colon cancer antigen 10 (PDB ID: 2HQ6) determined by X-ray diffraction (resolution: 1.75 Å) from the RCSB Protein Data Bank (https:// www. rcsb. org/) and used it as a target in our molecular docking experiments 28 . We prepared and optimized the structure of the receptor protein using MOE's ligx function with default settings. We drew the structures of the identified compounds in Chemdarw 17.0.0.206 in MOL format and performed 3D protonation, partial energy correction, and energy minimization using Merck molecular force field (MMFF94x). We determined the receptor active sites using MOE's site finder tool. We carried out the molecular docking analysis to predict the receptor-ligand interaction using flexible ligand-fixed receptor docking parameters with Triangle Matcher placement (scoring: London dG; retain: 30) and force field refinement (rescoring: London dG; retain: 10). We selected the most stable protein-ligand interactions based on their S-score minimum energy and root mean square deviation (RMSD). We recorded the docking score, RSMD, and 2D and 3D interactions 29,30 .

Results and discussion
Phytochemical screening. We carried out phytochemical screening on H. ramosissimum to identify the different chemical classes of the total methanolic extract active constituents using different reagents. The preliminary screening revealed the presence of alkaloids, terpenoids, saponins, tannins, flavonoids, coumarins, polyphenolics, and reducing sugars.
Total phenolic and flavonoid contents. We estimated the total phenolic content in the methanolic extract of H. ramosissimum to be 179.74 ± 0.58 µg/mL, in gallic acid equivalents. The equation for the standard curve was Y = 0.0031x − 0.0564 (Fig. S1), with R 2 = 0.9961. Moreover, we measured a total flavonoid content of 53.18 ± 0.60 µg/mL, in rutin equivalents. The equation for the standard curve was Y = 0.0032x + 0.0398 (Fig. S2), with R 2 = 0.9981.

Metabolomic analysis. GC-MS analysis of the lipoidal matter.
We performed a GC-MS analysis of the plant's lipoidal matter to address the lack of information regarding its phytoconstituents. The GC-MS chromatogram (Fig. S3) S4).

LC-ESI-MS/MS profiling.
We identified the compounds of the H. ramosissimum methanolic extract by LC-MS/MS. We compared their retention time (R t ), mass, and MS2 with standards-reported literature, and databases (TMIC and MassBank). In total, we tentatively identified 32 compounds, including alkaloids, flavonoids, coumarins, phenolic acids, and their derivatives. We identified 17 compounds in negative mode and 15 in posi- Free radical scavenging activity. The DPPH spectrophotometric assay is one of the most reliable and widely used methods to estimate the free radical scavenging effect of different plant extracts. The percent of inhibition values in the initial screening step of the two concentrations, namely, 1000 and 100 µg/mL, were 80.26 ± 1.50 (˃ 50) and 6.50 ± 0.73 (˂ 50), respectively. We serially diluted the extract that exceeded 50% inhibition (1000 µg/ mL) to provide five concentrations that we tested to determine that the IC 50 was 414.30 µg/mL (Fig. S9). For reference, Trolox has an IC 50 of 24.42 ± 0.87 µM. The H. ramosissimum methanolic extract showed a potent radicalscavenging effect, which may be attributed to its total phenolic and flavonoid contents.
The optical microscope staining images (Fig. 5) show the results of the SRB cytotoxicity assay against Colo-205 cell line of both total Colo-205 cells for two H. ramosissimum methanolic extract concentrations (0.01 and 100 µg/ mL), doxorubicin at the same concentrations, and negative control. The figure clearly shows that at 0.01 µg/mL, neither the extract nor doxorubicin caused significant morphological changes. Meanwhile, significant changes occurred at 100 µg/mL, confirming the dose-dependent character of the cytotoxicity of the extract. To investigate the safety of the H. ramosissimum methanolic extract on normal cells and the selectivity of its cytotoxicity on cancer cells, we performed the cytotoxic activity assay on OECs. The viability of OECs treated with 10 µg/ mL methanolic extract was 98.94% ± 0.77%. Meanwhile, this concentration showed a potent cytotoxic effect on Colo-205 cells by reducing cell viability to 75.95% ± 0.81%.  www.nature.com/scientificreports/ To further investigate the mechanism responsible for the cytotoxic activity of the H. ramosissimum methanolic extract, we carried out an apoptosis assay on Colo-205 cells. We used Annexin V-FITC/PI double staining and evaluated the apoptotic rates using flow cytometry. As illustrated in Fig. 6, the apoptotic rate was 56.14% in the positive control group using doxorubicin, whereas treating the cells with 18 µg/mL of methanolic extract (which is equal to its IC 50 ) significantly increased the apoptotic rate to 71.76%. These results suggest that the extract reduced Colo205 cell viability by inducing apoptosis.
To examine whether the cytotoxicity of the H. ramosissimum methanolic extract on Colo-205 was associated with cell cycle arrest, we conducted a PI-metric cell cycle analysis using flow cytometry (Fig. 7). The cell cycle histograms revealed that cells treated with the extract (18 µg/mL) had a markedly higher sub-G1 population (63.01%) than those treated with the positive control, paclitaxel (37.33%). This suggests that the extract induced apoptosis and/or necrosis in Colo-205 cells.
We reviewed the literature on the cytotoxic activity of different Heliotropium species and found that the n-hexane fraction of the ethanolic extract of H. subulatum aerial parts displayed cytotoxic activity at 3 mg/mL 91 . An in vitro assay against MRC5 human cells revealed that the methanolic extract of H. zeylanicum aerial parts exerted significant cytotoxicity with an IC 50 of 13 µg/mL 92 . Furthermore, the methanolic extract of the dried roots of H. indicum showed different mortality rates at different concentrations, with a lethal concentration (LC 50 ) of 47.86 μg/mL and LC 90 of 75.85 μg/mL in a brine shrimp lethality bioassay 93 . A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the whole plant ethanolic extract of H. indicum had significant antiproliferative activity against SKBR-3 human breast adenocarcinoma cells. The IC 50 of the extract was 34 ± 9.09 μg/mL, and that of the standard drug paclitaxel was 22.20 ± 2.30 μg/mL 94 . These findings suggest that the H. ramosissimum methanolic extract exerts a potent cytotoxic effect, especially against colorectal carcinoma, by inducing apoptosis. These results suggest that this extract is a promising candidate for the treatment of this type of cancer.

Molecular docking.
In silico molecular docking, techniques have enhanced drug discovery and development by allowing the structure-based exploration of ligand-receptor interactions. The cytotoxicity assays www.nature.com/scientificreports/ revealed that the cytotoxicity of the methanolic extract was more potent against colon cancer than against the other cell lines. We investigated the possible interactions between the 32 phytochemical compounds identified by LC-ESI-MS/MS and the colon cancer antigen 10 binding sites and compared them to the standard anticancer drug doxorubicin. Table 5 (Fig. 8). Orientin formed interactions with Ser73AA (H-donor) and π-H interaction with Glu 76 (Fig. 9).
Kaempferol (Fig. 10) formed hydrogen bonds with Gly75AA (H-donor), and taxifolin (Fig. 11) interacted with Gly75AA and Ser73AA (H-donor). The cytotoxicity of the tested extract may be attributed to the phenolic compounds and flavonoids with the best binding affinity in the molecular docking study.
The previous experimental studies are matched with our molecular docking results. Chlorogenic acid was reported to exhibit potential effects on cytotoxicity and inhibited human colon cancer cell proliferation through cell-cycle arrest and apoptosis 95 . In addition, orientin exhibited remarkable cytotoxicity and antiproliferative activity against HT-29 colon cancer cells, induced G0/G1 cell cycle arrest, regulated cyclin and cyclin-dependent protein kinases, and mediated apoptosis in human colorectal cancer HT-29 cells 96 . Further, taxifolin showed human colorectal cancer cell growth arrest in the G2 phase of the cell cycle and apoptosis in a concentrationdependent approach 97 . Besides, kaempferol's cytotoxic effects and induction of apoptosis in different human colorectal cancer cell lines have been reported 98 . www.nature.com/scientificreports/

Conclusion
In conclusion, the phytochemical screening of the total methanolic extract of the aerial parts of H. ramosissimum (Lehm.) DC. revealed the presence of alkaloids, terpenoids, saponins, tannins, flavonoids, coumarins, polyphenolics, and reducing sugars. It is worthy to mention that all the identified compounds in the n-hexane fraction are the first to be reported in the genus Heliotropium, in addition, thirty-two compounds of different chemical classes were tentatively identified by LC-ESI-MS/MS analysis. Besides, our findings revealed that the total methanolic extract of H. ramosissimum aerial parts exhibits a potent in vitro antitumor effect against several cell lines especially colorectal carcinoma (Colo-205) in a dose-dependent manner and with selectivity in comparison to normal cell lines (OEC). The methanolic extract reduces the viability of invasive Colo-205 cell lines by inducing apoptosis and/or necrotic cell death. Furthermore, a docking study for the identified compounds from the LC-ESI-MS/MS analysis to find out the candidates responsible for the cytotoxic activity where isochlorogenic acid and orientin showed the highest binding scores with colon cancer antigen 10. Our findings can aid in the creation of a new alternative candidate for the selective treatment of early stages of colorectal cancer with safety on normal cells. Future studies are needed to isolate the active constituent/constituents responsible for this potent cytotoxic activity.